From 13C-lignin to 13C-mycelium: Agaricus bisporus uses polymeric lignin as a carbon source

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO2. Recent research, however, demonstrated fungal conversion of 13C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated 13C-labeled lignin served as substrate for Agaricus bisporus, the world’s most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in 13C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in 13C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of 13C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin’s metabolic value for fungi.

Supplementary Method 2: Structural characterization of lignin by 1 H- 13 C HSQC NMR HSQC NMR spectra were recorded at 25 °C on a Bruker AVANCE III 600 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 5 mm cryo-probe located at MAGNEFY (MAGNEtic resonance research FacilitY, Wageningen, The Netherlands) and based on previously reported procedures (18).Purified soluble (LS), and water-insoluble (LR) residual lignin from 13 CLG+Ab and 13 CLG (0.25 mg) were dissolved in 0.6 mL DMSO-d6 and transferred to NMR tubes, though the water-insoluble fungal-treated samples were not completely soluble.The spectra were recorded using the "hsqcetgpsisp2.2"pulse program.In the 1 H dimension a spectral width of 12 ppm, an offset of 4.7 ppm, and 2048 increments were used, and in the 13 C dimension a spectral width of 200 ppm, an offset of 100 ppm, and 400 increments were used.Sixteen scans were recorded by using a recycle delay (D1) of 0.86 s, and a 1 JCH of 145 Hz.Spectra were processed using Gaussian apodization (GM; GB = 0.001, LB = −0.2Hz) in 1 H and a squared sine function (QSIN; SSB = 2) with 1024 increments in both 1 H and the 13 C dimensions.
The solvent peak (DMSO-d6) was used as an internal chemical shift reference (δC 39.5 ppm; δH 2.49 ppm) for processing Bruker TopSpin v4.0.5 was used.Phase correction was done manually and baseline correction was done automatically.Correlation peaks were assigned by comparison with literature (40,49,50,51,52).Semi-quantitative analysis of the HSQC volume integrals was performed according to Del Río (18).S2,6, G2 and H2,6 signals were used for S, G and H units, respectively, where S and H integrals were halved.The oxidized analogues were semi-quantified in a similar manner.Tricin, pCA and FA were similarly semi-quantified from their respective T2′,6′, pCA2,6 and FA2 signals.The β-O-4′ substructures were semi-quantified by their Cβ-Hβ correlations, whereas β-5 and β-β substructures were semi-quantified on their Cα-Hα correlations Volume integrals for resinol substructures were halved.Dihydroxypropiovanillone and dihydroxypropiosyringone (DHPV/S), hydroxypropiovanillone and dihydroxypropiosyringone (HPV/S), were semi-quantified from their Cβ-Hβ correlations.Benzaldehydes and cinnamaldehydes were quantified on their Cα-Hα correlations and cinnamyl alcohol was quantified on Cγ-Hγ correlations.Volume integrations were performed at similar contour levels, and between spectra the contour levels were normalized to an equal size of -OCH3 integral.Substructures were expressed per 100 aromatic rings (H + G + Gox + S + Sox).Supplementary Method 3: Sugar content and composition of the lignin isolates before and after fungal treatment For the quantification of (released) monosaccharides an ICS-5000 HPLC system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac PA1 guard column (2 mm ID ×50 mm) and a CarboPac PA-1 column (2 mm ID ×250 mm; both from Dionex) was used for analysis.The detection of the eluted monosaccharides was performed by an ED40 EC-detector running in the PAD mode (Dionex).10 μL of the 50 times diluted hydrolysates (LR and initial substrate), or nonhydrolyzed soluble fractions (LS) were injected on the system.Mobile phases used to elute the compounds were kept under nitrogen, and the column temperature was set at 20 °C.A flow rate of 0.4 mLmin -1 was used with the following elution profile of 0.  CLg+Ab (e, f).The harvested fungal biomass was collected in sieves (g), and lyophilized (h).

Fig. S2
Scanning electron microscope (SEM) pictures of A. bisporus grown on lignin.Lignin is visible as 'particles' on top of the fungal hyphae.

Fig. S3
Aromatic regions of 1 H- 13 C HSQC NMR spectra of water soluble (LS) and insoluble (LR) residual lignin after fungal treatment of biological duplicates ( 13 CLg+Agaricus bisporus I, 13 CLg+ Agaricus bisporus II,) compared with the control ( 13 CLG).Colour codes of spectra correspond to structures Figure 3 in main text and grey represents unassigned spectra.See supporting information for tables containing semiquantitative analysis of volume integrals (Supplementary Table 7).For explanation of sample codes see Figure 1 in main text.

Fig. S4
Aliphatic regions of 1 H- 13 C HSQC NMR spectra of water soluble (LS) and insoluble (LR) residual lignin after fungal treatment of biological duplicates ( 13 CLG+Agaricus bisporus I, 13 CLg + Agaricus bisporus II,) compared with the control ( 13 CLG).Colour codes of spectra correspond to structures Figure 3 in main text and grey represents unassigned spectra.See supporting information for tables containing semiquantitative analysis of volume integrals (Supplementary Table 7).For explanation of sample codes see Figure 1 in main text.C glucose grown Agaricus bisporus (b), 13 C lignin grown A. bisporus (c) and 12 C lignin spiked with 13 C carbohydrates and lipids grown A. bisporus (d).

Table S3
Agaricus bisporus carbon use efficiencies (CUEs) of various lignin isolates obtained from different sources, lignin purity, carbon, and fermentation parameters used.The fermentations were set-up as described in the main manuscript.NA = not analysed.

Table S4
Lignin content, analyzed by py-GC-MS, and relative abundance of pyrolysis products of the water insoluble lignin fractions (LR) corrected for RRF (18).See figure 1 for fractionation scheme and sample codes.Sum on the bases of previous structural classification (18,40,55)

Table S5
Lignin content, analyzed by py-GC-MS, and relative abundance of pyrolysis products of the water soluble lignin fractions (LS) corrected for RRF (18) See figure 1 for fractionation scheme and sample codes.Sum on the bases of previous structural classification (18,40,55)

Table S6
Lignin content, analyzed by py-GC-MS, and relative abundance of pyrolysis products of the lignin incorporated in the fungal biomass (FBM) corrected for RRF (18) See figure 1 for fractionation scheme and sample codes.Sum on the bases of previous structural classification (18,40,55)

Table S8
Fatty acid composition and content (based on dry matter) of Agaricus bisporus fungal biomass cultivated on different substrates (see Figure 1 for codes), and fatty acid content and composition of the 13 C lignin isolate ( 13 CLG).

Table S9
Fatty acid methyl esters, ergosterol and selected fragments with chemical formulas and exact masses.The screened m/z of 13 C isotopomers of the selected fragments (F) or parent (P) were used for integration to determine the 13 C fractional enrichment with mass precision of 10 ppm.

Table S10
13 C fractional labelling of fungal biomass compounds.Fatty acids, carbohydrates, amino acids, of A. bisporus (Ab) fungal biomass from 12 CG+Ab, 12 CLg+Ab, 13 CG+Ab, 12 C*Lg+Ab, and 13 CLg+Ab.All results are the average of biological duplicates, and error bars represent the standard deviations.Intracellularly, the C can further be converted and anabolized to new compounds (e.g., three carbon compounds).These newly formed compounds have are different mass isotopomers.The 13 C carbon can be implemented in the de novo biosynthesized compounds at different positions (positional isotopomers).Abundance of individual mass isotopomers is screened.Normalization of mass isotopomers abundances result in mass isotopomer distribution (MID).Further calculation steps involve the correction of the natural abundance of 13 C and then the summed fractional labelling can be calculated with the correction matrix.These calculations are explicitly explained in literature (42,43,44,56). .

Fig. S5
Fig. S5Chemical structures of fatty acid methyl esters and ergosterol with exact masses (Orbitrap MS) with indicated fragmentations and m/z that were selected for fractional labelling.
(H+G+Gox+S+Sox=100) b H2,6 integrals corrected for phenylalanine peak (PHE3,5) c Relative distribution of total interunit linkages in parenthesis d Relative volume integral of substructure versus volume integral of (H+G+Gox+S+Sox) e Absolute and relative in brackets of total β-O-4 linkages based on β signal f Ratio of Aβ(S/G-S)erythro and Aβ(S/G-S)threo; diastereomers for β-O-4′ aryl ethers coupled to G-units were not resolved.

Fig. S7 13 C
Fig. S7 13 C fractional labelling of fungal biomass compounds.Fatty acids and ergosterol (a), amino acids (b), carbohydrates (c), of A. bisporus (Ab) fungal biomass from 12 CG+Ab (light blue bars;glucose used as carbon source), from13 CG+Ab (dark blue bars;13 C-glucose used as carbon source), from12 CLg+Ab (grey bars; 12 C-lignin used as carbon source).All results are the average of biological duplicates, and error bars represent the standard deviations.The graphs on the right side indicate maximal labelling possible, calculated from fungal biomass increase and mycelium seed used, for13 CG+Ab (dark blue).

Fig. S8 Schematic explanation 13 C
Fig. S8Schematic explanation13 C fractional labelling of fungal biomass compounds.C present in the treatment consist of 12 C (fungal inoculum and debris from inoculum) and13 C (naturally occurring, and13 C-lignin substrate).Fungus metabolizes C to CO2 (extracellular and intracellular).Intracellularly, the C can further be converted and anabolized to new compounds (e.g., three carbon compounds).These newly formed compounds have are different mass isotopomers.The 13 C carbon can be implemented in the de novo biosynthesized compounds at different positions (positional isotopomers).Abundance of individual mass isotopomers is screened.Normalization of mass isotopomers abundances result in mass isotopomer distribution (MID).Further calculation steps involve the correction of the natural abundance of13 C and then the summed fractional labelling can be calculated with the correction matrix.These calculations are explicitly explained in literature(42,43,44,56). .
. Average and standard deviation were derived from technical duplicates.I and II indicate two biological replicates.
. Average and standard deviation were derived from technical duplicates.I and II indicate two biological replicates.

.
Average and standard deviation were derived from technical duplicates.I and II indicate two biological replicates.